1. Technical Field
The present invention relates generally to anti-VEGF Receptor 2 (VEGFR2; aka kinase insert domain-containing receptor, or KDR) antibodies, compositions and methods of using same. Such antibodies are useful, for example, in methods for treating and inhibiting a variety of disorders including age-related macular degeneration (AMD), diabetes and ischemic retinopathies, rheumatoid arthritis, psoriasis and a variety of oncological diseases including renal cell carcinoma, metastatic gastric or gastro-esophageal junction adenocarcinoma, breast cancer, hepatocellular carcinoma, colorectal cancer, prostate cancer, non-small cell lung cancer, ovarian cancers, melanoma, and recurrent glioblastoma multiforme, leukemias and solid tumors.
2. Description of the Related Art
VEGFR2/KDR is the primary angiogenic receptor and binds VEGF isoforms A, C, D and E, and is important for endothelial cell differentiation, as well as the mitogenic, angiogenic and permeability-enhancing effects of VEGF. An anti-KDR antibody may prevent all known VEGF isoforms from binding to VEGFR2/KDR and initiating signaling. In addition, because tumors secrete many more molecules of VEGF while the number of receptors remains relatively constant, targeting the receptor increases the probability of completely suppressing signaling even in the presence of very high levels of VEGF isoforms.
Angiogenesis
Angiogenesis, the formation of new blood vessels from existing vasculature, is a tightly regulated event and plays an important role in normal physiology such as embryonic development, follicular growth, wound healing, as well as in pathological conditions such as tumor growth and progression (1, 2).
Growth and metastasis of primary tumors is dependent on formation of new blood vessels. In the absence of neovascularization, tumors become necrotic or apoptotic and/or fail to grow beyond 2-3 mm3 in size (3). Tumor angiogenesis involves several processes, including endothelial cell activation, proliferation, migration, and tissue infiltration from preexisting blood vessels that are triggered by specific angiogenic growth factors produced by tumor cells and the surrounding stroma (1-4).
VEGF and VEGF Receptors
Several growth factors have been identified as possible regulators of angiogenesis (5). Among these factors, vascular endothelial growth factor (VEGF) and its receptors have been shown to play a key role in tumor angiogenesis (6-9).
VEGF is a homodimeric 34-42 kDa heparin-binding glycoprotein with potent angiogenic, mitogenic, and vascular permeability-enhancing activities (10, 11). VEGF regulates vasculogenesis during embryonic development and angiogenic processes during adult life (12, 13). VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGFs bind to and mediate activity through the VEGF receptors (VEGFRs). There are 3 VEGFRs including VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR) and VEGFR3 (14-16).
The physiological importance of VEGF and the VEGF receptors in blood vessel formation has been clearly demonstrated in gene knockout experiments (17-18). The VEGFR1 tyrosine kinase exhibits all the conserved motifs that are required for kinase activity. However, the level of phosphorylation of VEGFR1 in response to VEGF-A is low (37, 38). The function of VEGFR-1 is less well defined, it may act as a dummy/decoy receptor to sequester VEGF from VEGFR-2 binding and modulate VEGFR-2 signaling. It has been shown that VEGFR-3 may mediate lymphangiogenesis in response to VEGF-C and VEGF-D. VEGFR2/KDR is the primary angiogenic receptor and binds VEGF isoforms A, C, D and E, and is important for endothelial cell differentiation and mitogenesis.
Structure and Biology of KDR
The VEGFRs are receptor tyrosine kinases and belong to the same family of receptors as the PDGFs and fibroblast growth factors (FGFs). VEGFR2/KDR is a 200 kDa glycoprotein that consists of 7 Ig-like loops in the extracellular domain, a transmembrane domain, and two intracellular tyrosine kinase domains split by a kinase insert. The second and third Ig-like loops are high-affinity ligand-binding domains for VEGF while the first and fourth Ig-like loops regulate ligand binding and receptor dimerization, respectively. VEGF binds KDR with a Kd of 75-250 pM as compared to a Kd of 25 pM for VEGFR1.
KDR is primarily expressed on the cell surface of vascular endothelial cells. KDR is also found on the cell surface of hematopoietic cells, vascular smooth muscle cells (VSMCs), and some malignant cells.
KDR is the primary receptor in developmental angiogenesis and hematopoiesis and is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF. VEGFR2−/− knockout mice showed embryonic lethality at E8.5-9.5 with defective blood-island formation and vasculogenesis (41). Physiologically, the binding of VEGF to KDR results in endothelial cell activation, proliferation, migration, invasion and survival. Upon binding to VEGF, KDR receptors dimerize, leading to activation of kinase domains and transduction of KDR receptor signaling. Like many other receptor tyrosine kinases, the major intracellular signaling pathways that lead to angiogenesis include MAPK and PI3 kinase activation.
KDR as Molecular Target for Antibody Therapy
VEGFR2/VEGF axis is a predominant pathway in tumor angiogenesis. Numerous studies have shown that overexpression of VEGF and KDR are strongly associated with invasion and metastasis in human malignancies (6). VEGF receptors have been implicated in angiogenesis that occurs in many human solid tumors, including bladder (21), breast (22, 23), colon (24, 25), gastrointestinal (26), glioma (12, 27), renal (28), melanoma (29), and neuroblastoma (30).
The important role for KDR in tumor angiogenesis was directly demonstrated in studies in which expression of a dominant-negative KDR receptor resulted in decreased endothelial cell mitogenesis and growth inhibition of subcutaneous glioma tumors in athymic mice (31). Other studies confirmed this role using neutralizing soluble VEGF receptor (34, 35) as well as using KDR inhibitors including small molecule tyrosine kinase inhibitor (TKI) and KDR-specific Abs.
In addition to its effect on tumor angiogenesis, KDR is also found on some tumor cells such as leukemia cells and may directly mediate tumorigenesis through an autocrine loop that stimulates leukemia growth (42, 43).
Inhibition of KDR signaling can reduce angiogenesis and retards tumor growth (35, 36). The vast majority of current treatments targeting KDR are small-molecule tyrosine kinase inhibitors (TKIs). TKIs interfere with the binding of ATP or other substrates to the tyrosine kinases and disrupt the kinase catalytic activity. All of the TKIs developed to date (like Sunitinib) bind reversibly to the ATP binding site of the KDR kinase domain.
VEGF is expressed at high levels in various types of tumors (12, 19), and newly sprouting capillaries are clustered around VEGF-producing tumor cells (12). VEGF expression is strongly up-regulated under hypoxic conditions, such as those associated with rapidly growing tumors (20). Neutralizing VEGF by an antibody such as Avastin® (33, 34) is a clinically approved therapy to inhibit cancer or other angiogenesis diseases such AMD. However, resistance to VEGF blockade has been found even when given in combination with chemotherapy. This resistance may be associated with remodeled vasculature and with increased expression of other angiogenic factors. As such, there remains a need in the art for improved compositions and methods for inhibiting cancer and other diseases associated with angiogenesis.
Although both TKI and anti-KDR antibodies can inhibit KDR-mediated angiogenesis, the antibody approach has advantages over TKIs. In contrast to TKIs, an anti-KDR antibody is a more specific KDR targeting agent (i.e., it does not inhibit other VEGF receptors). Because of its high specificity, an anti-KDR antibody may be able to limit and/or avoid the off-target effects and toxicities caused by the less specific TKIs (44).
VEGF is expressed at high levels in various types of tumors (12, 19), and newly sprouting capillaries are clustered around VEGF-producing tumor cells (12). VEGF expression is strongly up-regulated under hypoxic conditions, such as those associated with rapidly growing tumors (20). Neutralizing VEGF by an antibody such as Avastin (33, 34) is a clinically approved therapy to inhibit cancer or other angiogenesis diseases such AMD. However, resistance to VEGF blockade has been found even when given in combination with chemotherapy. This resistance may be associated with remodeled vasculature and with increased expression of other angiogenic factors.
In contrast to Avastin® which binds one of the ligands (VEGF-A) only, an anti-KDR antibody is expected to prevent all known VEGFs from binding to VEGFR2/KDR. This may have a more profound inhibitory effect on tumor angiogenesis than just blocking VEGF-A. It is possible that therapy with an anti-KDR antibody may be effective in cases of Avastin® resistance. The potential advantage of targeting KDR over VEGF is summarized in Table 1.
TABLE 1Advantages of antibody therapy targeting KDR over VEGFAdvantages over anti-VEGFantibodiesMechanism/ReasonsFewer antibodies are needed to1) Overexpression/abundant production of VEGF inachieve effective inhibition of tumorsVEGF-KDR pathway2) KDR expression on endothelial cells is more constant than VEGF from tumor and cancer-related stromaSpecifically blocks KDR pathway An anti-KDR antibody selectively blocks KDRfrom activation by several VEGFsignalling from activation by related ligandsligands(VEGF-A, E, C, D)Provides an option forCombination therapy may overcome the acquired combination therapy with anti-resistance by anti-VEGF therapyVEGF for more efficient inhibition
Therefore, an antibody that targets KDR and blocks KDR signaling may have higher specificity and more complete target inhibition and therefore may have broad applications in both solid and liquid tumors as well as have the potential to overcome Avastin® resistance.
Anti-KDR Therapeutic Antibodies in Development
Ramucirumab (IMC-1121B)
Ramucirumab, which is being developed by ImClone Systems/Eli Lilly, is a fully human IgG1 mAb that binds human KDR (KD X50 pM) and blocks VEGF binding, thus inhibiting angiogenesis. Because Ramucirumab does not cross react with mouse KDR, a surrogate anti-mouse KDR antibody (DC-101) was generated and used for POC preclinical studies.
In phase I clinical trials in patients with advanced cancers, ramucirumab was well tolerated on weekly dosing schedules. Mechanism-related dose limiting toxicities were hypertension and deep vein thrombosis. Data from a phase II trial as a monotherapy in patients with metastatic renal cell carcinoma following KDR tyrosine kinase inhibitor therapy was reported recently (39). Patients with progressive disease or intolerance to either sorafenib, sunitinib or both were administered 8 mg/kg ramucirumab IV biweekly. Tumor assessments were performed every six weeks. A total of 40 patients were enrolled and 39 were treated. Nineteen patients (49%) had stable disease that lasted for more than 5 months; preliminary median progression free survival was 6 months. More phase II trials are ongoing in combination with dacarbazine in melanoma, with mitoxantrone/prednisone in patients with prostate cancer, with carboplatin/paclitaxel in patients with NSCLC and with oxaliplatin/folinic acid/5-fluorouracil in patients with colorectal cancer.
Ramucirumab is currently being evaluated in patients with breast cancer, gastric cancer or gastroesophageal junction adenocarcinoma and hepatocellular carcinoma in 3 Phase III studies. Three additional Phase 3 studies of ramucirumab with or without paclitaxel in metastatic gastric adenocarcinoma, in second line metastatic colorectal cancer and in second line non-small cell lung cancer are ongoing.
33C3
33C3 developed by AstraZeneca is a fully human anti-KDR antibody generated using XenoMouse™ technology. 33C3 binds the Ig domains 4-7 of KDR, and so has no impact on VEGF-A binding to KDR. It does not compete with antibody that interacts at the ligand binding site. 33C3 has high affinity for KDR (KD<1 nM) and inhibits VEGF-A induced phosphorylation of KDR. In vitro, 33C3 potently inhibits both tube length and number of branch points in a 2D angiogenesis assay and endothelial tube formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model (40). 33C3 is now at the early preclinical stage of development. 33C3 has not been tested in in vivo tumor models, due to lack of cross reactivity to mouse KDR.
TTAC-0001
TTAC-0001, a fully human anti-KDR antibody generated by phage display, is now in the preclinical stage of developed by PharmAbcine. The antibody showed potent anti-angiogenic efficacy against various cancer mouse models (45).